Pilot Study In Effects Of Nao Yi-AN(NYA) On Transplanted Neural Stem Cells In Rats With Intracerebral Hemorrhage In Basal Ganglion

Backgroud and purpose: Intracerebral hemorrhage(ICH) is a severe type of acute cerebrovascular disease with high morbidity and mortality. Finding effecacious medicines and investigating their mechanisms are very important. But present basic&clinic researches in ICH are focused on reducing neural loss from the injury to ameliorate behavioral deficit. In 1992, BA Reynold and S Weiss reported isolation of hippocampal neural stem cell from adult mice for the first time, which made many researchers see the promising future for curing such series of diseases in central nervous system. But most related progress was intended to transplant NSCs for replacement or gene therapy, much of which was only phenomena description, left many basic questions to answer, and in which many ethical problems needed solving, therefor, it will take a very long time and much effort to apply NSC in clinical practice. At the same time, accumulating evidences showed there are many phenomena of neurogenesis and endogenous NSC activation in many kinds of acute or chronic brain injury, which made it possible for the strategy of regulating the niche in injured brains and maneuvering endogenous NSC for repair. NYA is a Tradtional Chinese Medical drug for ICH at the acute phase, whose effects and mechanisms were verified in many clinical & basic experiments, in addition to which it showed positive effects on survival, proliferation and differentiation of NSC in vitro in pre-experiments. The experiment is intended to observe changes of endogenous or transplanted NSC in ICH rat brain and pssible effects of NYA on them.Methods: Sprague-Dawley rats were randomly divided into four groups including normal control group (n=5), sham control group (n=5), model control group (n=25) and NYA-treated group(n=25). By injecting collagenase type VII stereotaxically into right globus pallidus (1.40mm posterior and 3.20mm lateral to bregma and 5.60 ventral to the cortical surface), intracerebral hemorrhagic model was made. NYA was orally administered at dose of 4.92g/kg/d. At 1d, 3h, 6d, 13d, 27d, animals wererandomly chosen to receive intraperitoneal injectons of bromodeoxyuridine (BrdU 50mg/kg once per 4h during 8h , three times altogther), which were to be perfused transcardically the next day. The brains were sectioned at -18℃.Changes in distributions and numbers of NSC specific marker nestin positive cells and BrdU labeled newborn nuclears and effects of NYA were investigated from 2d to 28d folloing ICH; Acceding to the in vivo experiment above, cultured NSCs from neonatal rat hippocampus were transfected with pEGFP-Nl mediated with Lipofectamine?2000 or labeled with 1 U M BrdU, which were then transplanted into right subventricular zone (1.60mm anterior and 1.20mm lateral to bregma and 4.00mm ventral to the cortical surface). 2 days later , ICH was induced by the standard method. At 7d, animals were sacrificed to investigate distribution of EGFP-expressing or BrdU-labeled cells around hemotoma.Results: Gross histopatholoev: Compared with those of model control group, at 4d,swelling of leisoned lateral hemiphere of NYA-treated group was alleviated much more, at 7d,the size of hematoma was reduced more, and at 28d, the leisoned site restored better and had less effects on surrounding tissue.lt indicated that NYA can enhance absorption of brain edema and hemotoma,alleviate degenerationand necrosis in brain ,and quicken the repair. Changes of NSC and BrdU-labeled newborn nuclear: Most of Nestin-positive or BrdU-Labeled cells were located at the 2 sites: basal ganglion around hemotoma , ependyma and nearby subventricular zone (SVZ). No positive cell for the 2 markers in the 2 sites were found in normal control group and sham group , as well as in non-leisoned parenchyma , all the hippocampus and olfactory bulb in ICH animals. Nestin+ cells had 4 kinds of morphology,and BrdU+ nuclears were polymorphologic. Postive cell counting around hemotoma showed: at 2d, Nestin+ cells were seen around hemotoma in model group , the number of which increased at 4d,7d...